1. Technical Field
The present invention relates to a cDNA synthesis method.
2. Related Art
There has been reported by Boom et al. a method for more easily extracting nucleic acids from a biomaterial using a nucleic acid-binding solid-phase carrier such as silica particles and a chaotropic agent (see J. Clin. Microbiol., vol. 28, No. 3, pp. 495-503 (1990)). A method for extracting nucleic acids using a nucleic acid-binding solid-phase carrier such as silica and a chaotropic agent by adsorbing nucleic acids on the carrier including this method of Boom et al. mainly includes the following three steps: (1) a step of adsorbing nucleic acids on a nucleic acid-binding solid-phase carrier in the presence of a chaotropic agent (adsorption step); (2) a step of washing the carrier having the nucleic acids adsorbed thereon with a washing solution for removing nonspecifically bound contaminants and the chaotropic agent (washing step); and a step of eluting the nucleic acids from the carrier using water or a low salt concentration buffer (elution step). Here, as the washing solution used in the step (2), water or a low salt concentration buffer containing a water-soluble organic solvent, particularly ethanol in an amount of about 50 to 80% has been used for dissolving the chaotropic agent and also preventing the elution of the nucleic acids from the carrier.
However, when this water-soluble organic solvent remains in the step (3), an enzymatic reaction is inhibited when the extracted liquid is treated with an enzyme. Therefore, generally, after washing with an aqueous solution containing ethanol, a procedure is performed such that washing with 100% ethanol or a solvent having a higher volatility such as acetone is performed as needed, followed by drying so as to completely remove the organic solvent from the system. However, this procedure is known to have a problem that it takes a lot of time for this drying, and also ethanol may remain if the drying time is not sufficient, and in extreme cases, the nucleic acids are dried out and solidified, and therefore, it becomes difficult to elute the nucleic acids, resulting in a decrease in the yield of nucleic acids or a deterioration in reproducibility. Accordingly, the use of an organic solvent as described above has a problem that it is difficult to check the degree of dryness, and also an organic solvent such as ethanol or acetone is flammable and volatile, and therefore, particularly in the case of assuming that the operation is automated, it is also considered that there is a risk of fire or the like.
Accordingly, a method in which after nucleic acids are adsorbed on the carrier, the carrier is washed with water or a low salt concentration buffer which contains no organic solvents such as ethanol in the washing step (2), and the nucleic acids are eluted with water or a low salt concentration buffer at 50 to 70° C. in the elution step (3), whereby ribonucleic acids (RNA) are extracted has been developed (see JP-A-11-146783).